Amino-acid functionalized porous silicon nanoparticles for the delivery of pDNA

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Amino-acid functionalized porous silicon nanoparticles for the delivery of pDNA

Arnaud Chaix, Eduardo Cueto-Diaz, Anthony Delalande, Nikola Knezevic, Patrick Midoux, Jean-Olivier Durand, Chantal Pichon, Frédérique Cunin

To cite this version:

Arnaud Chaix, Eduardo Cueto-Diaz, Anthony Delalande, Nikola Knezevic, Patrick Midoux, et al..

Amino-acid functionalized porous silicon nanoparticles for the delivery of pDNA. RSC Advances,

Royal Society of Chemistry, 2019, 9 (55), pp.31895-31899. �10.1039/c9ra05461h�. �hal-02338857�

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Amino-acid functionalized porous silicon nanoparticles for the delivery of pDNA†

Arnaud Chaix,‡

^a

Eduardo Cueto-Diaz,‡

^a

Anthony Delalande,‡

^b

Nikola Knezevic,

^c

Patrick Midoux,

^b

Jean-Olivier Durand,

^a

Chantal Pichon§

^b

and Frederique Cunin § *

^a

Porous silicon nanoparticles as a novel platform in gene therapy, have shown to be an e ffi cient vehicle for the delivery of nucleic acids in cells. For the fi rst time, a family of porous silicon nanoparticles has been produced featuring an amino-acid functionalized cationic external surface aiming at pDNA complexation. The amino acid-based pDNA nanocarriers, displaying an average diameter of 295 nm, succeeded in transfection of HEK293 cells with an e ffi ciency 300 times superior to “ bare ” porous silicon nanoparticles.

Introduction

Gene therapy is a promising approach for the treatment of carcinogenic cells as it aims to deliver genetic materials into specic cells to produce a therapeutic eﬀect, by either altering or correcting an abnormality or by promoting a new cell func- tion. However, one of the main obstacles for the clinical appli- cation of this therapy aimed at malignant tumors is the lack of safe and tumor-selective gene vectors.

¹

The ideal gene carrier should provide efficient gene delivery properties, specic cell targeting, avoid inammatory/immunogenic responses in the exposed area, and must be easily manufacturable and clinically applicable. In this regard, virus-based gene vectors, although exhibiting good transfection efficiency, yield important side effects. Multiple signicant problems, such as inammatory responses, random integration in host genome and toxicity using recombinant viral vectors have been reported.

²

Alternative routes have been investigated leading to the development of viral-free systems. Non-viral vectors reported in the literature are synthetic chemical systems including cationic liposomes

³

and polymers.

⁴

In addition physical methods such as gene gun,

⁵

electroporation,

⁶

particle bombardment,

⁷

and ultrasound

⁸

are alternative methods for the direct injection of nucleic acids.

Despite the low eﬃciency of non viral vectors compared to viral

ones, their cost, availability, high modulability (no size limita- tions), with scarce generation of immunologic and no onco- genic adverse eﬀects make them an attractive alternative for gene delivery.

^9,10

Porous silicon materials and nanoparticles have proved to bear a great potential for nanomedicine applications, owing to their excellent in vivo biocompatibility and biodegradability.

^11–16

The major degradation by-product is silicic acid, which appears to be non-toxic to human cells.

^9,10

Herein, we report the rst approach on porous silicon nanoparticles bearing positively charged amino-acids motifs (histidine, lysine) to electrostati- cally interact and transport the long polyanion plasmid DNA (pDNA) to cancer cells. The fusogenic feature of histidine resi- dues at mild acidic pH, as that of endosomes, will yield the escape of the nanocomplexes from endosomes, once internal- ized inside cells, and favor cell transfection.

¹⁷

Porous silicon nanoparticles (pSiNp) were prepared by anodic etching of boron doped crystalline silicon in a solution of aqueous hydrouoric acid (HF) in ethanol following procedure previously pub- lished.

¹⁸

Typically the pSiNp were produced aer liing-oﬀ the porous lm using an electropolishing current, ultrasonication treatment, and centrifugation. The obtained pSiNp exhibited a hydrodynamic diameter of 190 nm with a polydispersity of 0.2 in accordance with transmission electron microscopy images (Fig. S1, ESI†). Three diﬀerent formulations of pSiNp were prepared, two of them containing either the amino-acids lysine or histidine, and the other one bearing a primary amine in the periphery, as a control for simple positively charged surface.

¹⁹

In this study, the pSiNp surface was rst silanized with APTES followed by the peptide coupling of the amino acids (histidine and lysine) prior to pDNA complexation (Scheme 1). Silaniza- tion of pSiNp with 3-aminopropyltriethoxysilane (APTES) con- ducted at room temperature over 18 hours, provided the free amino functional group (pSiNp@NH

₂

) allowing easy further

a

Institut Charles Gerhardt Montpellier, Charles Gerhardt Montpellier, Universit´e de Montpellier, UMR 5253 CNRS-ENSCM-UM2-UM1, 2 Place Eug`ene Bataillon, 34095 Montpellier Cedex 05, France. E-mail: frederique.cunin@enscm.fr

b

Centre de Biophysique Mol´eculaire in Orleans (CBM), UPR4301, France

c

Biosense Institute, University of Novi Sad, Dr Zorana Djindjica 1, 21000 Novi Sad, Serbia

† Electronic supplementary information (ESI) available. See DOI:

10.1039/c9ra05461h

‡ Contributed equally to this work.

§ Co-last authors.

Cite this: RSC Adv., 2019, 9, 31895

Received 16th July 2019 Accepted 24th September 2019 DOI: 10.1039/c9ra05461h rsc.li/rsc-advances

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functionalization (Scheme 2). The graing of the amine moiety was conrmed by zeta potential measurements and DLS with an increase of the surface charge from 26.1 mV to +50.2 mV due

to the presence of ammonium groups, and with an increase of the dynamic diameter from 190 nm to 295 nm respectively (Table 1, Fig. S2 and S3, ESI†). Although the DLS method is not suitable for non-spherical objects such as pSiNps, it was used in the present study in order to provide an estimation of the pSiNps size.

The dramatic increase in the hydrodynamic diameter of the intermediate formulation pSiNp@NH

₂

was ascribed to solvation provoked by the presence of the amine groups (Table 1). In addi- tion APTES can polymerize and form a multilayer at the surface of the nanoparticles thus increase their hydrodynamic diameter.

Finally, this increase of the hydrodynamic diameter could also result from the fact that porous silicon nanoparticles are neither spherical nor isotropic 3D nanoparticles. Attenuated total reec- tance Fourier-transform infrared (ATR-FTIR) spectroscopy, as pre- sented in Fig. S4, ESI,† also conrmed anchoring of the aminated function. The quantication of the attached APTES moiety was determined by elemental analysis (2.0% (N), 10.4% (C)) with a graed amount of 1.16 mmol (APTES) per gram of pSiNp (Fig. S5, ESI†). Aer silanization, the two protected amino acid motifs were condense through peptide coupling. Benzotriazol-1-yl-oxy- tripyrrolidinophosphonium hexauorophosphate (PyBOP) was the selected coupling reagent as it promotes rapid binding and yields less noxious by-products; N,N-diisopropylethylamine (DIPEA) was chosen as the organic base.

Next, Boc and Fmoc were successively removed by means of TFA and piperidine addition, leading to the nal formulations, pSiNp@Lys and pSiNp@His. The supernatant in both cases was collected for dosing the histidine and lysine and then for quantication of the histidine and lysine immobilized on the pSiNp. The reaction progress was monitored by zeta potential measurement, DLS and FTIR spectroscopy. Zeta potential measurement of pSiNp@Lys and pSiNp@His was carried out aer sonication of the samples for 90 s to minimize Scheme 1 Schematic representation of the amino-acid functionalized porous silicon nanoparticles, with: (a) lysine, (b) histidine, and (c) NH

2

.

Scheme 2 Reaction scheme for the chemical functionalization of the pSiNp with histidine and lysine, and for the complexation of pDNA: (a) APTES, toluene, 50

C 20 h; (b) Fmoc-Hist(Boc) $ OH, PyBOP, DIPEA, EtOH, rt, 18 h, CH

2

Cl

2

, TFA, 18 h; (c) Boc-Lys(Fmoc), PyBOP, DIPEA, EtOH, rt, 18 h, CH

2

Cl

2

, TFA,18 h.

Table 1 Surface charge, size and quanti ﬁ cation of pSiNp@amino acid in EtOH at 25

C

Formulations

z -potential (mV)

m g

mg

¹

pSiNp Size (nm)

pSiNp 26 1 — 190

pSiNp@NH

2

50 1 — 295

pSiNp@His 47 2 7 295

pSiNp@Lys 45 5 84 295

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aggregation. The resulting values at +45 mV and +47 mV respectively indicated successful immobilization and depro- tection of the amino acids at the surface of the nanoparticles (Table 1 and Fig. S2, ESI†). The hydrodynamic diameter of both formulations resulted in a similar value of 295 nm (Table 1 and Fig. S3, ESI†). The FTIR spectra revealed two characteristic bands at 790 cm

¹

and 800 cm

¹

attributed to the bending vibrations of primary and secondary N–H bonds present in the three formulations, but absent in the pSiNp, conrming the successful functionalization of the pSiNp (Fig. S4, ESI†). In addition, the presence of intense narrow bands at 1050 cm

¹

was ascribed to n

s

(Si–O–Si) conrming the APTES graing into the silicon nanoparticles. Deprotection and Fmoc removal were then conducted in a two steps sequence. First, the removal of the acidic proton at the 9-position of the uorene ring system by a mild base, (secondary cyclic amine (piperidine)) was per- formed, and secondly the subsequent b-elimination was con- ducted yielding a highly reactive species such as dibenzofulvene, and intermediate that were immediately trap- ped by the organic base forming the stable adduct (for details, see ESI†). Fmoc-aminoacid assay performed by means of UV-Vis spectroscopic experiments at 290 nm, allowed the quantica- tion of each attached amino acid, thus indicating the graing of 0.045 mmol (histidine) and 0.575 mmol (lysine) per mg of pSiNp (more details in Fig. S5 and S6, ESI†). The diﬀerent graing eﬃciency was ascribed to the high steric hindrance and low solubility of the histidine moiety.

The complexation eﬃciency of plasmid DNA was monitored at physiological pH (7.4) for the three formulations pSiNp@NH

2

, pSiNp@Lys and pSiNp@His by gel shi experi- ments (Fig. 1). No complexation was observed between the non- functionalized pSiNp and pDNA whatever the ratio used. As pSiNp exhibits a surface charge of – 26 mV due to the presence of silanols at their surface, they did not show aﬃnity for nega- tively charged pDNA. In contrast pSiNp@NH

₂

, pSiNp@Lys and pSiNp@His with surface charge of +50 mV, +45 mV, +47 mV respectively, eﬃciently complexed pDNA. For most of SiNp derivatives, the complexation started to form at 1/1 and then full complexation was observed at a ratio of 1/5 (Fig. 1).

The transfection eﬃciency of all formulations pSiNp@NH

2

, pSiNp@Lys and pSiNp@His at 1/1, 1/5 and 1/10 ratios was investigated on HEK 293 human cells using pDNA encoding luciferase reporter gene expression as a read out (Fig. 2). Except for pSiNp, all formulations did result into eﬃcient gene trans- fer. The level of gene expression is dependent on the ratio for pSiNp@NH

₂

and pSiNp@His. For pSiNp@Lys formulation, there is no clear signicant diﬀerence of luciferase units whatever the ratio used. For the others, the transfection eﬃ- ciency was higher at a ratio of 1/10, the values were close to 10

⁷

RLU per mg proteins (Fig. 2A). We assessed the eﬀect of chlo- roquine, known as endosomolytic agent reported to improve drastically the cell transfection.

^20,21

The transfection eﬃciency was highly improved at least by 10-fold as seen for pSiNp and by 100-fold for pSiNp@NH

2

, pSiNp@His and pSiNp@Lys compared to transfection made in absence of chloroquine (Fig. 2B). These data inform that the endosomal escape was not so eﬃcient even when pSiNp was substituted with histidine,

known to induce efficient endosomal escape via proton sponge like effect. This behaviour is quite different to that observed for cationic polymers or liposomes, which become more efficient once substituted with histidine residues.

²²

Note that this improvement could be also due to another reported eﬀect of chloroquine on the dissociation of pDNA complexes.

²³

Indeed, too tight complexation of DNA with the vector could hamper the recognition by the transcription machinery reducing the gene expression.

The cell uptake eﬃciency was evaluated by ow cytometry using formulations made with uorescein-labelled pDNA Fig. 1 Gel retardation assay demonstrating DNA@nanoparticle complexation. A constant amount of pDNA (1 m g) was complexed with nanoparticles at nine di ﬀ erent ratios in HEPES (10 mM, pH 7.4).

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(Fig. 3). The mean uorescence intensity (MFI) measured is related to the amount of pDNA. The surface and intracellular associated uorescence intensities (Fig. 3, white and grey bars, respectively) were evaluated thanks to the well-known quench- ing of uorescein label with Trypan blue. PSiNp formulation was hardly taken up by cells compared to other formulations, which explains the low transfection eﬃciency. In contrast, the intracellular associated uorescence intensities were at least 3- fold more than the surface associated one for pSiNp@NH

2

, pSiNp@His and pSiNp@Lys revealing that they were eﬃciently uptaken. The uptake was formulation and ratio-dependent. At low ratio (1/3), the uptake capacity of diﬀerent formulations could be ranked as follows: pSiNp@Lys > pSiNp@NH

2

> pSiN- p@His > pSiNp. When made at a higher ratio (1/10), the order

was diﬀerent: pSiNp@His > pSiNp@Lys ¼ pSiNp@NH

₂

> pSiNp.

To assess if internalized DNA formulations were present in acidic compartment, cells were treated with monensin in presence of a neutral phosphate buffer (PBS). As being an ionophore, monensin treatment can reverse the acidity of the milieu. Since the uorescein uorescence intensity is quenched in acidic medium, it can be restored at neutral pH. Data reveal that at low ratio, all complexes were not localized acidic compartments because no signicant differences of the MFI before and aer monensin treatment (Fig. 3, grey versus black bars). At ratio 1/10, similar observation can be made except for pSiNp@His, for which the MFI increased a bit though not signicantly. Last, cellular viability was also measured by XTT according to the manufacturer's instructions. In the absence of Fig. 2 Transfection e ffi ciency: luciferase activities (RLU per mg protein) measured 48 h following transfection of HEK 293 human cells with indicated silicon nanoparticles formulated with pDNA encoding luciferase gene at a ratios of 1/1, 1/5 and 1/10 (white bar, grey bar and black bar, respectively) in absence (A) or presence of 100 m M chloroquine (B).

Fig. 3 Cell uptake e ﬃ ciency: cells were incubated for 2 h at 37

C with indicated silicon nanoparticle formulated with 1 m g of fl uorescein-labelled pDNA either at ratio 1/3 (A) or 1/10 (B). Cell associated fl uorescence intensity was recorded before and after quenching with Trypan blue to get the surface associated (white bar) and intracellular-associated (grey bar) cell fl uorescence intensities. Black bars correspond to cell fl uorescence intensities recorded upon monensin treatment. Values are mean of cell fl uorescence intensities recorded by fl ow cytometry with 10 000 events.

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chloroquine, transfection made with all formulations used at ratio of 1/1 did not induce any cytotoxicity. At higher ratios, a low cytotoxicity (20%) was observed except for pSiNp@His formulation, which did not result in any reduction of cell viability (Fig. S7, ESI†). The substitution of porous silicon with histidine residues results in a preservation of cell viability as we observed for the drastic reduction of polyethyleneimine cyto- toxicity once it was substituted.

²⁴

In cells treated with chloro- quine, the toxicity (up to 40%) was higher likely due to the eﬀect of this treatment. Again, there is a distinction of pSiNp@His formulation, which did not lead to any viability alteration. By contrast, a substantial cytotoxicity was measured (40%) in the presence of chloroquine for pSiNp@NH

2

and pSiNp@Lys used at ratios of 1/3 and 1/10. This is in line with our previous conclusions.

Porous silicon nanoparticles were chemically modied bearing an amino acid functionalized cationic surface for pDNA complexation and cell transfection in vitro. The formulations of histidine-functionalized pSiNp gave very promising results for plasmid delivery. Transfection eﬃciency and uptake data indi- cate that, as any delivery vehicle, a higher ratio is required to complex and protect pDNA. We demonstrate that the amino- acid functionalized nanoparticles succeed in transfection of HEK cells, and that they are non-toxic to the cells 48 hours aer transfection. In this work, a new type of material for trans- fection and for plasmid delivery was explored. pSiNps did not generate adverse eﬀects and showed limited toxicity compared to usual cationic vectors made of polymers or liposomes.

Con ﬂ icts of interest

There are no conicts to declare.

Acknowledgements

The authors gratefully acknowledge the R´ egion Occitanie and the Fonds Europ´ eens de D´ eveloppement R´ egional (FEDER) for

nancial support.

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